The inhibition of dopamine reuptake via the dopamine transporter (DAT) has been characterized as the primary mechanism by which cocaine produces its psychomotor stimulant and reinforcing actions. In order to understand further the molecular mechanisms underlying cocaine abuse, structure-function studies have been directed toward characterizing the DAT protein at a molecular level. The design, synthesis and evaluation of 3-alpha-(diphenylmethoxy)tropane (benztropine) analogs have provided potent and selective probes for the DAT. Structure-activity relationships (SAR) have been developed that contrast with those described for cocaine, despite significant structural similarity. Furthermore, behavioral evaluation of many of the benztropine analogs, in animal models of cocaine abuse, has suggested that these two classes of tropane-based dopamine uptake inhibitors have distinct pharmacological profiles. In general, our previous studies have shown that the benztropine analogs, do not demonstrate efficacious locomotor stimulation in mice, do not fully substitute for a cocaine discriminative stimulus and are not appreciably self-administered in rats or nonhuman primates. These compounds are generally more potent than cocaine as dopamine uptake inhibitors, in vitro, although their actions in vivo are not consistent with this action. As such, we have described this class of compounds as atypical dopamine uptake inhibitors. By varying the structures of the parent compounds, a series of N-substituted benztropine analogues has been discovered that readily penetrates the blood brain barrier, but compared to cocaine, they typically have a slower onset and longer duration of action, which is a suitable profile for development as pharmacotherapeutics and may be directly related to their lack of cocaine-like behavioral profiles. In this regard, we have extended the studies on our previously characterized cocaine antagonist, JHW 007 (N-n-butyl-4, 4-diF-benztropine), and discovered that JHW 007 and structurally related benztropine analogues were not self administered, in rats and indeed could selectively attenuate self administration of cocaine while having no effect on food administration. These studies further demonstrate the lack of abuse liability for these agents and strengthen their potential for development as therapeutics. Studies using site-directed mutagenesis and molecular pharmacology have revealed differences in binding domains between the benztropines, cocaine and other structurally diverse dopamine uptake inhibitors. Interestingly, experimental evidence using the DAT inhibitors cocaine, WIN 35,428, and several benztropine analogues and comparing them to the substrates dopamine and MDMA has provided evidence, at the molecular level, of binding interaction differences that correlate with their distinctive behavioral profiles. Of note, cocaine binds to an outward facing conformation of the DAT, whereas the benztropines as well as the substrates, prefer an inward facing closed conformation. These conformational studies provide evidence at the molecular level that the atypical dopamine uptake inhibitors are indeed functioning differently than cocaine at the DAT and this is correlated with their distinct behavioral profiles. As the benztropine class of molecules has yet to be translated into clinical studies, we recently focused attention on modafinil, which binds to the DAT and is currently used clinically for the treatment of sleep disorders. Modafinil has been evaluated as a potential medication to treat methamphetamine and cocaine abuse and is also being used off-label for the treatment of ADHD. As modafinil has structural and pharmacological features that resemble the benztropines, we have embarked on an SAR study to further characterize these compounds at DAT, NET and SERT and to explore novel compounds with improved water solubility. Thus far these novel compounds demonstrate a unique SAR profile. Modifications to the modafinil template have resulted in molecules with high affinity (up to 1000-fold higher than the parent drug) and selective binding to the DAT. In addition, computational studies support experiments in the mutant DATs that suggest modafinil prefers an inward facing conformation of the DAT, like the benztropines. Metabolism, pharmacokinetic and behavioral analyses of selected ligands are underway that have identified potential lead compounds e.g., JJC8-016 for further development. Selected lead compounds show promising results in rodent models of both cocaine and methamphetamine abuse. Mechanistic studies are underway to elucidate how these novel DAT inhibitors block the reinforcing effects of these psychostimulants without significantly affecting dopamine levels in the Nucleus Accumbens, as measured by microdialysis and fast scanning cyclic voltammetry as well as electrophysiology. In addition, off target actions, including interactions at sigma1 receptors and dopamine D2 autoreceptors are being investigated. Of note, compounds with a dual DAT/sigma1 receptor profile may result in a superior therapeutic to treat psychostimulant use disorders.